Comparative host gene transcription by microarray analysis early after infection of the Huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229E

Abstract

The pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) at the cellular level is unclear. No human cell line was previously known to be susceptible to both SARS-CoV and other human coronaviruses. Huh7 cells were found to be susceptible to both SARS-CoV, associated with SARS, and human coronavirus 229E (HCoV-229E), usually associated with the common cold. Highly lytic and productive rates of infections within 48 h of inoculation were reproducible with both viruses. The early transcriptional profiles of host cell response to both types of infection at 2 and 4 h postinoculation were determined by using the Affymetrix HG-U133A microarray (about 22,000 genes). Much more perturbation of cellular gene transcription was observed after infection by SARS-CoV than after infection by HCoV-229E. Besides the upregulation of genes associated with apoptosis, which was exactly opposite to the previously reported effect of SARS-CoV in a colonic carcinoma cell line, genes related to inflammation, stress response, and procoagulation were also upregulated. These findings were confirmed by semiquantitative reverse transcription-PCR, reverse transcription-quantitative PCR for mRNA of genes, and immunoassays for some encoded proteins. These transcriptomal changes are compatible with the histological changes of pulmonary vasculitis and microvascular thrombosis in addition to the diffuse alveolar damage involving the pneumocytes.

FIG. 1.

Linkage of the altered genes by using Pathway Assist (version 2.53) shows clustering of genes related to apoptosis, inflammation, and coagulation. Only genes with known direct interactions are shown. The software is available from Ariadne Genomics Inc. +, upregulated/increased expression; −, downregulated/decreased expression.

FIG. 2.

Validation of microarray data by semiquantitative RT-PCR. The expression patterns of all nine selected genes and the housekeeping gene, which were assessed for their relative abundances in mock infection, at 2 h and 4 h after SARS-CoV inoculation, and at 2 h and 4 h after HCoV-229E inoculation, were consistent with the expression pattern shown in the microarray experiment.

FIG. 3.

Fold changes of expression of nine selected genes by RT-qPCR. The P values of the differences in fold changes of the target gene expressions are as follows: TFPI2 (SARS-CoV at 2 h versus HCoV-229E at 2 h), 0.02; TFPI2 (SARS-CoV at 4 h versus HCoV-229E at 4 h), 0.02; PAI1 (SARS-CoV at 2 h versus HCoV-229E at 2 h), 0.05; PAI1 (SARS-CoV at 4 h versus HCoV-229E at 4 h), 0.05; THBS1 (SARS-CoV at 2 h versus HCoV-229E at 2 h), 0.03; THBS1 (SARS-CoV at 4 h versus HCoV-229E at 4 h), 0.35; IL-8 (SARS-CoV at 2 h versus HCoV-229E at 2 h), <0.01; IL-8 (SARS-CoV at 4 h versus HCoV-229E at 4 h), <0.01; NFKB2 (SARS-CoV at 2 h versus HCoV-229E at 2 h), 0.05; NFKB2 (SARS-CoV at 4 h versus HCoV-229E at 4 h), <0.01; JUNB (SARS-CoV at 2 h versus HCoV-229E at 2 h), <0.01; JUNB (SARS-CoV at 4 h versus HCoV-229E at 4 h), 0.28; PHLDIA (SARS-CoV at 2 h versus HCoV-229E at 2 h), 0.20; PHLDIA (SARS-CoV at 4 h versus HCoV-229E at 4 h), <0.01; CARD10 (SARS-CoV at 2 h versus HCoV-229E at 2 h), 0.03; CARD10 (SARS-CoV at 4 h versus HCoV-229E at 4 h), 0.04; BAX (SARS-CoV at 2 h versus HCoV-229E at 2 h), 0.04; BAX (SARS-CoV at 4 h versus HCoV-229E at 4 h), 0.24.

FIG. 4.

Validation of protein expression for TFP1, PAI1, and IL-8 by enzyme immunoassay. The P values of the differences in protein expression are as follows: TFPI2 (SARS-CoV at 2 h versus HCoV-229E at 2 h), <0.01; TFPI2 (SARS-CoV at 4 h versus HCoV-229E at 4 h), 0.37; TFPI2 (SARS-CoV at 12 h versus HCoV-229E at 12 h), 0.18; TFPI2 (SARS-CoV at 24 h versus HCoV-229E at 24 h), 0.12; PAI1 (SARS-CoV at 2 h versus HCoV-229E at 2 h), <0.01; PAI1 (SARS-CoV at 4 h versus HCoV-229E at 4 h), <0.01; PAI1 (SARS-CoV at 12 h versus HCoV-229E at 12 h), <0.01; PAI1 (SARS-CoV 24 at h versus HCoV-229E at 24 h), 0.01; IL-8 (SARS-CoV at 2 h versus HCoV-229E at 2 h), <0.01; IL-8 (SARS-CoV at 4 h versus HCoV-229E at 4 h), <0.01; IL-8 (SARS-CoV at 12 h versus HCoV-229E at 12 h), <0.01; IL-8 (SARS-CoV at 24 h versus HCoV-229E at 24 h), <0.01).